Vaccinia virus polypeptides

ABSTRACT

This document provides methods and materials related to polypeptides present in a vaccinia virus (e.g., polypeptides that can be isolated from naturally processed and presented class I polypeptides originating from vaccinia virus, a member of the Orthopoxvirus family). For example, methods for generating a vaccine comprising one or more of vaccinia virus polypeptides disclosed herein for preventing or treating Orthopoxvirus infection are provided. In addition, kits related to the use of vaccinia polypeptides are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/379,311, filed Sep. 1, 2010. The disclosure of the priorapplication is considered part of (and is incorporated by reference in)the disclosure of this application.

BACKGROUND

1. Technical Field

This document provides methods and materials relating to isolatedvaccinia virus-derived polypeptides. For example, this document relatesto specific and naturally processed and HLA presented vacciniavirus-derived polypeptides isolated from vaccinia virus, a member of theOrthopoxvirus family. This document provides methods for generating avaccine for preventing or treating Orthopoxvirus infection that inducesa protective therapeutic immune response. The vaccines can include oneor more of the isolated vaccinia virus polypeptides provided herein. Inaddition, this document provides kits related to the use of vacciniavirus polypeptides.

2. Background Information

Polypeptide-based vaccines use small polypeptide sequences derived fromtarget proteins as epitopes to provoke an immune reaction. Thesevaccines are a result of an improved understanding of the molecularbasis of epitope recognition, thereby permitting the development ofrationally designed, epitope-specific vaccines based on motifsdemonstrated to bind to human class I (HLA I) or class II (HLA II) majorhistocompatibility complex (MHC) molecules. Of particular interest hasbeen the discovery of epitopes that are specifically recognized by Tcells for prophylaxis and treatment of infectious diseases.

Over the centuries, naturally occurring smallpox, with its case-fatalityrate of 30 percent or more and its ability to spread in any climate andseason, has been universally feared as one of the most devastating ofall the infectious diseases. The use of vaccinia virus as a vaccineenabled the global eradication of naturally occurring smallpox. The lastnaturally occurring case of smallpox occurred in Somalia in 1977. In May1980, the World Health Assembly certified that the world was free ofnaturally occurring smallpox. Routine vaccination against smallpox inthe United States ended in 1971, and except for some soldiers andlaboratory workers, no one has been vaccinated since 1983. However,terrorist activities in the early 21st century as well as importedoutbreaks of monkeypox (a member of the Orthopox virus family) in theUSA, spurred renewed interest in biodefense countermeasures for thesepublic health threats (Artenstein et al., Expert Rev. Vaccines,7:1225-1237 (2008) and Giulio et al., Lancet Infect. Dis., 4:15-25(2004)).

SUMMARY

This document provides methods and materials related to vaccinia viruspolypeptides. For example, this document provides vaccinia viruspolypeptides that have the ability to be naturally processed andpresented by HLA molecules. This document also provides methods andmaterials (e.g., vaccines) for preventing or treating Orthopoxvirusinfections. For example, the vaccines provided herein can include one ormore of the vaccinia virus polypeptides provided herein and can have theability to induce a protective therapeutic immune response within amammal (e.g., a human). In addition, this document provides kits relatedto the use of vaccinia virus polypeptides.

As described herein, two-dimensional liquid chromatography coupled tomass spectrometry was used to identify 116 vaccinia virus polypeptides,encoded by 61 open reading frames, from a human B-cell line (homozygousfor HLA class I A*0201, B*1501, and C*03) after infection with vacciniavirus (Dryvax). The identification of these naturally processed andpresented polypeptides resulting from vaccinia virus infection can beused to aid in understanding the immune process and can be used togenerate vaccines against Orthopoxviruses.

In general, one aspect of this document features an isolatedpolypeptide, wherein the amino acid sequence of the polypeptide is asset forth in any one of SEQ ID NOs:1-83.

In another aspect, this document features a composition comprising atleast one isolated polypeptide selected from the group consisting of SEQID NOs:1-82 and 83 and at least one polypeptide selected from the groupconsisting of SEQ ID NOs:84-115 and 116. The composition can furthercomprise an adjuvant.

In another aspect, this document features a method of preventing ortreating variola virus infection in a subject. The method comprises, orconsists essentially of, administering to the subject a compositioncomprising an adjuvant and at least one polypeptide, wherein the aminoacid sequence of the polypeptide is as set forth in any one of SEQ IDNOs:1-83. The subject can be a human.

In another aspect, this document features a vaccine comprising, orconsisting essentially of, at least one isolated polypeptide, whereinthe amino acid sequence of the polypeptide is as set forth in any one ofSEQ ID NOs:1-83. The vaccine can comprise at least one polypeptideselected from the group consisting of SEQ ID NOs:84-115 and 116. Thevaccine can comprise an adjuvant.

In another aspect, this document features a method of enhancing theimmune response in a subject to a vaccine. The method comprises, orconsists essentially of, administering an agent capable of increasingthe expression of a transporter associated with antigen processing inthe subject, wherein the vaccine comprises at least one isolatedpolypeptide, wherein the amino acid sequence of the polypeptide is asset forth in any one of SEQ ID NOs:1-83. The transporter can be TAP1,TAP2, or Tapasin.

In another aspect, this document features a method of inducing an immuneresponse against at least one isolated polypeptide selected from thegroup consisting of SEQ ID NOs:1-82 and 83. The method comprises, orconsists essentially of, administering the polypeptide to a subject inan amount effective to induce an immune response against thepolypeptide. The polypeptide can be administered in combination with apolypeptide selected from the group consisting of SEQ ID NOs: 84-115 and116. The polypeptide can be administered in combination with apharmaceutically acceptable excipient, carrier, diluent, or vehicle. Themethod can comprise administering to the subject an agent capable ofincreasing expression of a TAP molecule. The immune response can be acell mediated immune response. The cell mediated immune response can bea cell mediated cytolytic immune response. The cell mediated immuneresponse can be a class I-restricted T cell response.

In another aspect, this document features a kit comprising, orconsisting essentially of, (a) at least one polypeptide selected fromthe group consisting of SEQ ID NOs:1-82 and 83, and (b) an adjuvant. Thekit can comprise at least two polypeptides selected from the group. Thekit can comprise at least one polypeptide selected from the groupconsisting of SEQ ID NOs:84-115 and 116.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram of a protocol for isolating and identifying HLAclass I polypeptides from B cells infected with vaccinia virus.

FIG. 2 contains graphs plotting the distribution of HLA polypeptideamino acid length for (A) vaccinia virus polypeptides and (B) allidentified polypeptides, and the putative sorting of polypeptides bybinding allele for (C) vaccinia virus polypeptides and (D) allidentified polypeptides. Polypeptides were classified by allele usingtheir C-terminal amino acid: L or V assigned to A*0201, F or Y assignedto B*1501, and all other polypeptides marked as NA (not assigned).

FIG. 3A is a graph of the MS/MS spectrum of the vaccinia viruspolypeptide IQYPGSEIKGNAY (SEQ ID NO:16) found in SCX fractions 4 and 5,as annotated by Scaffold, including mass accuracy of the precursor massin parts-per-million (ppm). A table of the fragment ions matched and theexperimental error of the fragment ions is included. The Orbitrap surveyspectrum of the precursor ion is shown. FIG. 3B is a graph containingthe same information for the vaccinia virus polypeptide IQYPGSKIKGNAY(SEQ ID NO:17) as identified from SCX fractions 14-16. Note thedifferent precursor mass, as well as concomitant changes to fragment ionmasses consistent with the change in amino acid E (Glu) to K (Lys).

FIG. 4 is a pie graph of vaccinia epitopes directly identified by MS/MSthat are classified by predicted HLA-binding strength as determined bythe netMHC algorithm at the Center for Biological Sequence Analysis,Technical University of Denmark, (“http” colon, slash, slash “www” dot“cbs.dtu.dk” slash “services” slash “NetMHC” slash). Polypeptidesequences with calculated IC₅₀ values <50 nM were classified as strongbinding, IC₅₀ values between 50 nM and 500 nM were classified as weakbinding, and IC₅₀>500 nM were classified as non-binding polypeptides.

DETAILED DESCRIPTION

This document provides methods and materials related to vaccinia viruspolypeptides. For example, this document provides vaccinia viruspolypeptides that have the ability to be naturally processed andpresented by HLA molecules. This document also provides methods andmaterials (e.g., vaccines) for preventing or treating Orthopoxvirusinfections. For example, the vaccines provided herein can include one ormore of the vaccinia virus polypeptides provided herein and can have theability to induce a protective therapeutic immune response within amammal (e.g., a human). In addition, this document provides kits relatedto the use of vaccinia virus polypeptides.

This document provides compositions (e.g., vaccine compositions)containing one or more vaccinia virus polypeptides provided herein. Insome cases, a vaccinia virus polypeptide provided herein can have theability to be naturally processed and presented by a class I MHCmolecule. Examples of such vaccinia virus polypeptide provided hereininclude, without limitation, those vaccinia virus polypeptides set forthin SEQ ID NOs:1-83 of Table 2. In some cases, the polypeptides set forthin SEQ ID NOs:1-83 can be used individually or as a mixture for theprevention and/or therapeutic treatment of Orthopoxvirus infections invitro and in vivo, and/or for improved diagnostic detection ofOrthopoxvirus infections. Any appropriate combination of thepolypeptides listed in Table 2 can be used. For example, the combinationcan include at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, ormore polypeptides selected from Table 2. For example, the polypeptidescorresponding to SEQ ID NOs: 1-10 can be used in any combination. Insome cases, the polypeptides corresponding to SEQ ID NOs:1-10 and SEQ IDNOs:70-83 can be used in any combination. For example, the polypeptidescorresponding to SEQ ID NO:1 and SEQ ID NO:3 can be used in anycombination with SEQ ID NOs:70-83. In some cases, a combination of thepolypeptides listed in Table 2 can be used with the exception of 2, 3,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more polypeptides selectedfrom Table 2. For example, the polypeptides corresponding to SEQ IDNOs:1-10 can be used in any combination with the exception of SEQ IDNOs:11-83. For example, the polypeptides corresponding to SEQ IDNOs:1-83 can be used in any combination with the exception of SEQ IDNO:10, SEQ ID NO:20 and SEQ ID NO:30.

In some cases, one or more of the polypeptides set forth in SEQ IDNOs:1-83 can be used in combination with at least one of thepolypeptides set forth in SEQ ID NOs:84-116 of Table 3. Any appropriatecombination of the polypeptides listed in Table 2 can be used with atleast one of the polypeptides set forth in SEQ ID NOs:84-116 of Table 3.In some cases, a combination can include at least 2, 3, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, or more polypeptides selected from Table 2 withat least one of the polypeptides set forth in Table 3. For example, thepolypeptides corresponding to SEQ ID NOs:1-10 from Table 2 can be usedin combination with SEQ ID NO:84 of Table 3. In some cases, thepolypeptides corresponding to SEQ ID NOs:1-10 and SEQ ID NOs:70-83 canbe used in combination with SEQ ID NO:84. In some cases, the combinationcan include at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, ormore polypeptides selected from Table 2 with at least 2, 3, 5, 10, 15,20, 25, or more polypeptides selected from Table 3. For example, thepolypeptides corresponding to SEQ ID NOs:1-10 can be used in combinationwith SEQ ID NOs:84-90. In some cases, SEQ ID NOs:1, 5, and 10 can beused in combination with SEQ ID NOs:85, 90, and 100. In some cases, oneor more vaccinia virus polypeptides set forth in SEQ ID NOs: 19, 29, and49 can be used in combination with one or more vaccinia viruspolypeptides set forth in SEQ ID NOs:41 and 42.

In some cases, a composition can be designed to include one or morevaccinia virus polypeptides that have a sequence present within avaccinia virus polypeptide that is expressed during an early phase of apoxvirus infection in combination with one or more vaccinia viruspolypeptides that have a sequence present within a vaccinia viruspolypeptide that is expressed during a late phase of a poxvirusinfection. For example, one or more vaccinia virus polypeptides setforth in Table 2 or Table 3 for ORFs A7L, D13L, D6R, DBL, E10R, E6R,E8R, G4L, H4L, H7R, and I1L (e.g., a polypeptide involved in a latephase of infection) can be used in combination with one or more vacciniavirus polypeptides set forth in Table 2 or Table 3 for ORFs A44L, A46R,A48R, A52R, A8R, B12R, B13R, B15R, B1R, C11R, C12L, C2L, E5R, E9L, F11L,F12L, F16L, F1L, H5R, J3R, J4R, J6R, K1L, K3L, K6L, K7R and N2L (e.g., apolypeptide involved in an early phase of infection). In some cases, oneor more vaccinia virus polypeptides set forth in SEQ ID NOs:19 (A44L)and 49 (E5R) can be used in combination with one or more vaccinia viruspolypeptides set forth in SEQ ID NOs:29 (A7L), 41 (D13L), and 42 (D13L).

In some cases, a composition can be designed to include two or morevaccinia virus polypeptides (e.g., two, three, four, five, six, seven,eight, nine, ten, or more vaccinia virus polypeptides) that potentiallyhave the ability to bind to class I MHC molecules with high bindingaffinity. For example, two or more vaccinia virus polypeptides set forthin SEQ ID NOs:1, 19, 29, 37, 41, 42, 44, 49, 64, and 68 can be used incombination to form a composition (e.g., a vaccine composition).

The polypeptides provided herein (e.g., the polypeptide presented inTables 2 and 3) can include oxidized amino acid residues (e.g., oxidizedforms of methionine) or can lack oxidized amino acid residues.

The term “isolated” refers to material which is substantially oressentially free from components which normally accompany the materialas it is found in its native state. Thus, isolated polypeptides asdescribed in this document do not contain materials normally associatedwith the polypeptides in their in situ environment. The term“polypeptide” generally refers to a short chain of amino acids linked bypolypeptide bonds. Typically, polypeptides comprise amino acid chains ofabout 2-100, more typically about 4-50, and most commonly about 6-20amino acids.

Any appropriate method can be used to obtain a vaccinia viruspolypeptide provided herein. For example, polypeptides having thesequence set forth in any one of SEQ ID NOs:1-116 can be synthesized bymethods known to one skilled in the art of making polypeptides. Ofcourse, other methods in the art would be appropriate. In some cases,simple chemical polypeptide synthesis techniques can be used to obtain avaccinia virus polypeptide provided herein. In some cases, apolynucleotide sequence encoding for a vaccinia virus polypeptide ofinterest can be inserted into a plasmid or other vector that can then bedelivered to hosts that can be induced to transcribe the polynucleotideinto the polypeptide of interest. In some cases, a polynucleotidesequence for a larger polypeptide can be inserted into host cells thatcan produce the larger polypeptide and then process that polypeptideinto a smaller polypeptide or a functionally equivalent variant ofinterest.

A composition provided herein containing one or more polypeptides setforth in SEQ ID NOs:1-83 of Table 2 or any appropriate combination ofpolypeptides as described herein can be formulated to provide apolypeptide-based vaccine. In some cases, such a vaccine can be designedto be based on a combination of naturally processed and presentedvaccinia virus polypeptides. For example, a polypeptide-based vaccinecan be designed to include at least one polypeptide selected from SEQ IDNOs:1-83 and at least one polypeptide selected from SEQ ID NOs:84-116.Any appropriate method can be used to formulate a polypeptide-basedvaccine including, for example, those methods used to formulatepolypeptide-based vaccines directed against other viral targets.Examples of polypeptide-based vaccines directed to other viral targetsare described elsewhere (see, e.g., Belyakov et al., Proc. Natl. Acad.Sci. U.S.A., 95(4):1709-1714 (1998) and Jackson et al., Proc. Natl.Acad. Sci. U.S.A., 101(43):15440-15445 (2004)). In some cases, a vaccinecomposition provided herein can include one or more polypeptides setforth in SEQ ID NOs:1-83 (or any appropriate combination of polypeptidesas described herein) in combination with the active ingredients orpolypeptides of a vaccine composition described elsewhere (see, e.g.,Belyakov et al., Proc. Natl. Acad. Sci. U.S.A., 95(4):1709-1714 (1998)and Jackson et al., Proc. Natl. Acad. Sci. U.S.A., 101(43):15440-15445(2004)). Such vaccine composition can provide a level of protectionagainst Orthopoxvirus infections as well as infections by the otherviral targets.

In some cases, a vaccine composition provided herein can be designed toprevent or treat an Orthopoxvirus infection. For example, a vaccinecomposition provided herein can have the ability to induce a protectiveor therapeutic immune response within a mammal (e.g., a human). In somecases, a vaccinia virus polypeptide provided herein can be a highlyconserved polypeptide across the family of Orthopoxvirus members. Insuch cases, a vaccine composition containing such a highly conservedpolypeptide can be used to provide protection against multiple membersof the Orthopoxvirus family. In some cases, a vaccine compositionprovided herein can be directed against any Orthopoxvirus. For example,a vaccine composition provided herein can be directed against monkeypox,cowpox, and camelpox. In some cases, a vaccine composition providedherein can be directed against vaccinia or variola major or minor. Theterm “vaccine” as used herein refers to immunogenic compositions thatare administered to a subject for the prevention, amelioration, ortreatment diseases, typically infectious diseases. In some cases, one ormore features of other vaccine preparations can be incorporated into avaccine composition provided herein. For example, a polypeptide used tocreate a vaccinia vaccine can be included within a vaccine compositionprovided herein. Examples of vaccinia-specific single polypeptidevaccines that have one or more features that can be included in themethods and materials (e.g., a vaccine composition) provided herein aredescribed elsewhere (see, e.g., Snyder et al., J. Virol., 78(13):7052-60(2004) and Drexler et al., Proc. Natl. Acad. Sci. U.S.A., 100(1):217-22(2003)).

A polypeptide provided herein (e.g., a polypeptide set forth in Table 2or Table 3) can be formulated into a vaccine composition using anyappropriate method. In some cases, a polypeptide provided herein can becombined with a pharmaceutically acceptable carrier or pharmaceuticalexcipient. The term “pharmaceutically acceptable” refers to a generallynon-toxic, inert, and/or physiologically compatible composition. A term“pharmaceutical excipient” includes materials such as adjuvants,carriers, pH-adjusting and buffering agents, tonicity adjusting agents,wetting agents, preservatives, and the like. Examples of adjuvantsinclude, without limitation, CpG, aluminum sulfate, aluminumphosphylate,and MF59. In some cases, vaccines or components of a vaccine can beconjugated to, for example, a polysaccharide or other molecule, toimprove stability or immunogenicity of one or more vaccine components.In some cases, a polypeptide provided herein (e.g., a polypeptide setforth in Table 2 or Table 3) can be formulated into a vaccinecomposition containing cells. For example, one or more polypeptidesprovided herein can be included within a cellular vaccine. Anyappropriate method can be used to prepare a cellular vaccine or thecomponents of a cellular vaccine.

The methods and materials provided herein can be used in combinationwith other techniques having the ability to enhance the immune responseof a vaccine. For example, a vaccine composition provided herein can bedesigned to include or to be used in combination with an effectiveamount of an agent that can augment the level of a TAP molecule and/or atapasin molecule within a cell. Increasing the level of TAP and/ortapasin molecules within a cell can increase the immunogenicity of avaccine composition containing a polypeptide provided herein. In somecases, the techniques described elsewhere (Vitalis et al., PLoS Pathog.,1(4):e36 (2005)) can be used in combination with the methods andmaterials provided herein. Examples of TAP molecules include, withoutlimitation, TAP-1 molecules and TAP-2 molecules. In some cases, aneffective amount of an agent that can augment the level of a TAP1molecule alone, a TAP2 molecule alone, both TAP-1 and TAP-2 molecules, atapasin molecule alone, or the combination of TAP-1, TAP-2, and tapasinmolecules can be used in combination with the methods and materialsprovided herein.

The levels of a TAP molecule can be augmented using agents that canincrease TAP expression including, without limitation, interferon-γ andp53. In some cases, the levels of a TAP molecule or a tapasin moleculecan be augmented by administering a nucleic acid molecule encoding a TAPmolecule or a tapasin molecule. The target cell can be any appropriatecell to which one wishes to generate an immune response. For example, ina prophylactic therapy or a vaccine, the target cell can be anessentially normal cell (e.g., a cell expressing normal TAP levels) thatmay not have been otherwise exposed to an antigen. In such a case, theagent that augments TAP can be co-administered with the antigen (e.g., apolypeptide provided herein) to which one wishes to generate an immuneresponse. For example, when used as a therapeutic, the target cell canbe previously infected with a pathogen (such as a virus or bacteria).

This document also provides methods and materials for treating mammals(e.g., humans) having an Orthopoxvirus infection. For example, acomposition provided herein can be administered to a mammal having anOrthopoxvirus infection under condition effective to reduce the severityof one or more symptoms of the Orthopoxvirus infection. Treatment ofindividuals having an Orthopoxvirus infection (e.g., a vaccinia virusinfection) can include the administration of a therapeutically effectiveamount of one or more polypeptides set forth in SEQ ID NOs:1-83. In somecases, treatment can include the use of one or more polypeptides setforth in SEQ ID NOs:1-83 individually or as a mixture. In some cases,one or more polypeptides set forth in SEQ ID NOs:1-83 can be used incombination with at least one of the polypeptides set forth in SEQ IDNOs:84-116. The polypeptides can be used or administered as a mixture,for example, in equal amounts, or individually, provided in sequence, oradministered all at once. The term “therapeutically effective amount”refers to that amount of the agent sufficient to result in ameliorationof symptoms, e.g., treatment, healing, prevention or amelioration ofsuch conditions. In providing a subject with a polypeptide providedherein (e.g., a vaccinia-derived polypeptide), combinations or fragmentsthereof, capable of inducing a therapeutic effect, the amount ofadministered agent will vary depending upon such factors as thesubject's age, weight, height, sex, general medical condition, previousmedical history, etc. In some cases, the amount of administered agentcan vary depending upon the subject's HLA allele type. The subject canbe, for example, a mammal. The mammal can be any type of mammalincluding, without limitation, a mouse, rat, dog, cat, horse, sheep,goat, cow, pig, monkey, or human.

This document also provides kits that can be used for a variety ofapplications including, without limitation, combining reagents necessaryfor producing vaccine compositions. Such vaccine compositions caninclude one or more polypeptides provided herein (e.g., one or morevaccinia-derived polypeptide described herein) as well as adjuvants,diluents, and pharmaceutically acceptable carriers. In some cases, a kitprovided herein can include a combination of vaccinia virus polypeptides(e.g., vaccinia-derived polypeptides) as described herein. In somecases, a kit provided herein can include at least 2, 3, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, or more polypeptides described herein. Anyappropriate combination of the polypeptides can be used. In some cases,a kit provided herein can include one or more adjuvants and can includeinstructions for preparing and administering a vaccine composition.

In some cases, a kit provided herein can be designed as a diagnostickit. For example, a kit provided herein can be designed to includereagents that can be used to detect cellular immune responses. In somecases, a kit provided herein can be designed to include polypeptidesthat can be used to detect antigen specific T cells. Such polypeptides(e.g., a polypeptide listed in Table 2 or 3) can be used to detectantigen specific T cells in samples from Orthopoxvirus (e.g., vacciniavirus) infected or exposed subjects. In some cases, such polypeptides(e.g., a polypeptide listed in Table 2 or 3) can be used to detectantigen specific T cells post-vaccination of a subject to determine theefficacy of immunization.

Any appropriate method can be used to detect antigen specific T cellsusing a polypeptide provided herein. For example, flow cytometry,enzyme-linked immunospot (ELISPOT), cytokine secretion, directcytotoxicity assays, and lymphoproliferation assays can be used todetect antigen specific T cells using a polypeptide provided herein. Insome cases, flow cytometry using MHC class I tetramers can be used,particularly for vaccinia epitope specific quantitation of CD8⁺ T cells.Such kits can include at least one polypeptide provided herein. In somecases, such a kit can include at least 2, 3, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, or more polypeptides provided herein for the detection ofantigen specific T cells.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Discovery of Naturally Processed and HLA-PresentedClass I Polypeptides from Vaccinia Virus Infection using MassSpectrometry for Vaccine Development Materials and Methods CellCulturing and Vaccinia Virus Infection

Epstein-Barr virus (EBV)-transformed B-cells (European Collection ofCell Cultures—ECACC, number 86052111, Salisbury, Wiltshire, UK),homozygous for the HLA-A*0201, B*1501, and C*03 were of human origin andused as antigen presenting cells (APCs). The New York City Board ofHealth (NYCBOH, Dryvax®) vaccine-strain of Vaccinia virus was culturedin HeLa cells in Dulbecco's modified Eagle's medium, supplemented with5% fetal calf serum (FCS; Life Technologies, Gaithersburg, Md.). B-cellswere infected with live Vaccinia virus at a multiplicity of infection(moi) of 0.1 PFU/cell for 2 hours and further maintained for 24-30 hoursin RPMI-1640 supplemented with 8% FCS. These uninfected andVaccinia-infected cells (approximately 1×10⁹ cells each) were used forobtaining cell lysates for the class I HLA molecules purification.

Isolation of HLA-Associated Polypeptides from Uninfected andVaccinia-Infected Cells

Class I HLA molecules were purified from human homozygous B-cells usingan immunoaffinity approach, and their associated polypeptides wereextracted as previously described (Slingluff et al., J. Immunol.,150:2955-2963 (1993) and Johnson et al., J. Am. Soc. Mass Spectrom.,16(11):1812-1817 (2005)). In brief, cells were lysed with a buffercontaining 20 mM Tris, pH 8.0, 150 mM NaCl, 1% CHAPS and proteaseinhibitors (1 mM Pefabloc SC, Roche Applied Science, Indianapolis,Ind.). The clarified supernatants were passed over a protein A-sepharose4B (Sigma) column containing the monoclonal antibody (mAb) W6/32specific for HLA-A, B and C (Parham et al., J. Immunol., 123(1):342-349(1979) and Hogan et al., Cancer Immunol. Immunother., 54(4):359-371(2005)). The HLA molecules (1.2 mg/mL) were dissociated from their boundclass I polypeptides using 0.2N acetic acid, pH 2.7, and polypeptideswere separated from the HLA by filtration through a 10-kDa molecularmass cutoff filter (Millipore, Bedford, Mass.).

Strong Cation Exchange Fractionation

Strong cation exchange (SCX) fractionation of the sample was performedafter desalting the polypeptide pool with a reversed phase 1 mm by 8 mmpolypeptide trap (Michrom BioResources, Auburn, Calif.). SCXchromatography used a gradient of 5 mM KH₂PO₄, pH 3.0 to 0.4 M KCl of 5mM KH₂PO₄, pH 3.0. Acetonitrile was added to each mobile phase to 20% byvolume. Desalted polypeptides were loaded onto a polysulfoethylaspartamide column (Michrom BioResources) at 0.5% mobile phase A, and agradient was developed to 20% B over 20 minutes at a flow rate of 200μL/min, then from 20% B to 80% B over the next 10 minutes. Two-minutefractions were collected; each fraction was vacuum centrifuged todryness before analyzing the fractions by nLC-MS/MS.

LTQ-Orbitrap nLC-MS/MS Analyses

Automated nLC-MS/MS analyses were performed on a commercial linear iontrap-Fourier transform hybrid mass spectrometer (LTQ-Orbitrap, ThermoFisher Scientific, Waltham, Mass.), interfaced to a nano-scale liquidchromatograph and autosampler (Eksigent NanoLC 1D, Dublin, Calif.),using a 15 cm by 75 μm i.d. column packed with Magic C_(18AQ) (5 μmparticles, 200 Å pore size, Michrom BioResources). The autosamplerloaded 5-20 μL onto a 0.25 μL pre-column (Optimize Technologies, OregonCity, Oreg.), custom-packed with Magic C₈, 5 μm, 200 Å (MichromBioResources). Mobile phase A consisted of water/acetonitrile/formicacid (98/2/0.2 by volume) and mobile phase B wasacetonitrile/water/formic acid (90/10/0.2 by volume). A 90 minute LCmethod employed a gradient from 2% to 40% B over 60 minutes, followed bya second segment to 90% B at 85 minutes, with a column flow of 0.4μL/minute. A third pump was used to load polypeptides from theautosampler to the pre-column, with 0.05% TFA and 0.15% formic acid inwater at 15 μL/minute.

SCX fractions were analyzed multiple times by nLC-MS/MS usingdata-dependent acquisition of tandem mass spectra. The first experimenttargeted singly charged precursors between 750 and 1500 on the m/z(molecular mass m divided by charge z) scale. A second experimenttargeted doubly and triply charged precursors between m/z 375 and 750,consistent with MHC class I polypeptides which are predominantly 9-11amino acids long.

The LTQ-Orbitrap was operated in a data-dependent mode, first acquiringan Orbitrap survey scan with 60,000 resolving power (FWHM at m/z 400), atarget cell population of 1×10⁶ ions, and a maximum ion fill time of 300ms. The preview Fourier transform was used to select the five mostabundant ions for MS/MS experiments in the LTQ. LTQ MS/MS spectra wereacquired with a 2.5 mass unit isolation width, target ion population of1×10⁴ ions, one microscan, maximum ionization fill time of 100 ms,normalized collision energy of 35%, activation Q of 0.25, and activationtime of 30 ms. Once ions were selected for MS/MS, they were subsequentlyexcluded for 45 seconds. The exclusion window was 1 m/z below, and 1.6m/z above the exclusion mass.

Polypeptide Identification from MS/MS Data

Database searching was performed using the SWIFT workflow tool developedin-house. SWIFT coordinates the generation of database search files(using extract_msn software from ThermoFisher Scientific), initiatesdatabase searches using MASCOT, Sequest, and X!Tandem search engines,and integrates these search results using Scaffold (Ver. 1_(—)07_(—)00,Proteome Software, Portland, Oreg.). Database searches were done againsta subset of the SwissProt database (January, 2007) obtained using theBioworks 3.2 (Thermo Fisher) database utility to select human, bovine,and vaccinia proteins. Bovine proteins were included in the databasesince cell cultures were supplemented with fetal calf serum. Databasesearches were performed with a precursor mass tolerance of 7parts-per-million (ppm), fragment ion mass tolerance of 0.6 mass units,and without any protease specificity. Single oxidation on methionineresidues was considered as a variable modification. The database wasappended with decoy protein entries consisting of randomized proteinsequences (MASCOT utility) for estimating the false-positive rateresulting in a database of 43,400 entries (including the randomizeddecoy entries). Results from all analyses of all SCX fractions werecombined by Scaffold and exported to an Excel spreadsheet.

The Scaffold program (Proteome Software) was used to combine searchresults from all of the LC-MS/MS analyses and to calculate polypeptideidentification probabilities using Scaffold's implementation ofPolypeptideProphet (Keller et al., Anal. Chem., 74(20):5383-5392 (2002)and Nesvizhskii et al., Anal. Chem., 75(17): 4646-4658 (2003)). Anexport function within Scaffold (Spectrum Report) was used to create atext file of all search results passing a lenient filter threshold of80% protein probability, with at least 1 polypeptide identified above an80% polypeptide probability threshold; 23,800 search results met thosecriteria, 1521 of these were from decoy polypeptides. Additionalfiltering was then applied to this dataset while estimating thefalse-positive rate (FPR) of identifications from the incidence ofidentifications from the decoy database using the formula: 2×# matchesto decoy polypeptides/(number of true positives+number of falsepositives) as described elsewhere (Elias et al., Nat. Methods,4(3):207-214 (2007)). The FPR was calculated as a function of thresholdsfor the following scoring parameters: Sequest cross-correlation score(XCorr), the difference between the top two normalized cross-correlationscores (ΔC_(n)), Mascot Ion Score, and mass error of the precursor mass.

Results HLA Class I Polypeptide Identification by Tandem MassSpectrometry

FIG. 1 provides an overview of the protocol used to sequence HLA class Ipolypeptides isolated from B-cells after infection with vaccinia.Sixteen strong cation exchange fractions were analyzed by nano-scaleliquid chromatography coupled with tandem mass spectrometry (nLC-MS/MS)on the LTQ-Orbitrap. Two data sets were acquired, each using multipleinjections as described above. The initial data set was acquired withexternal mass calibration, followed by a second data set collected withinternal mass calibration using lock masses at m/z 391.2843 and m/z445.1200 as described elsewhere (Olsen et al., Mol. Cell Proteomics,4(12):2010-2021 (2005)). These analyses generated 214,800 MS/MS spectrathat were searched against the human, bovine, and Vaccinia subset ofproteins in the SwissProt database (January 2007 version).

Polypeptide sequencing by mass spectrometry involves matching MS/MSfragmentation spectra against theoretical fragmentation spectracalculated for any polypeptide sequence in the database within atolerance window of the molecular weight of the polypeptide as measuredby the mass spectrometer. As a result, every database search will returna result, and each of these matches to a sequence must be evaluated fortheir validity. A variety of scoring metrics exist from which athreshold is established for accepting or rejecting the search resultfrom any MS/MS spectrum. The goal of that threshold is to minimize thenumber of incorrect or random matches that are accepted (falsepositives) while also trying to minimize the rejection of correctsequence matches (false negatives). Most of these scoring criteria havebeen developed within the context of identifying polypeptides generatedfrom cleavage of proteins with trypsin. Trypsin cleaves on theC-terminal side of arginine and lysine and this cleavage specificitygreatly reduces the list of candidate polypeptide sequences that arematched against the experimental spectra. The basic C-terminus oftryptic polypeptides provided by the Arg and Lys side chains, favorablydirects fragmentation during MS/MS, influencing the scores from thedatabase search results.

HLA class I polypeptides are not constrained to a basic C-terminal aminoacid. For most common alleles, hydrophobic amino acids predominate inthe C-terminal position, though basic residues such as Lys, Arg, Pro, orHis, are often found somewhere in the C-terminal half of thepolypeptide. Also not requiring Lys or Arg as the C-terminal amino acidfrom the database, greatly increases the number of candidate sequenceswhose theoretical fragmentation spectrum must be matched against theexperimental fragmentation spectrum. Because of these differences weimplemented the use of decoy database entries during the search asdescribed elsewhere (Elias et al., Nat. Methods, 4(3):207-214 (2007)).For each protein in the database an additional entry is created with itsamino acid sequence randomized, and labeled as such, in its accessionidentifier. During the database search these decoy proteins competeagainst authentic proteins for the best match to experimental spectra.Search results that identify polypeptides from a decoy protein are knownto be incorrect. The rate of matches to polypeptides from the decoyproteins is used to estimate the false-positive rate as described above(Polypeptide identification from MS/MS data).

Search results are summarized in Table 1 at the estimated 1% and 5% FPR.5915 MS/MS spectra were identified at the 1% FPR (30 matches againstdecoy polypeptides), representing 2731 unique sequences. 65 of thesepolypeptides were unique to vaccinia virus, originating from 44 vacciniavirus proteins. At the 5% FPR, the number of matched spectra increasedfrom 5915 to 12,819 (313 matches against decoy polypeptides), 5601 ofwhich were unique sequences. Of these 5601 unique sequences, 116polypeptides originated from 61 vaccinia proteins.

TABLE 1 Summary of polypeptides identified by two-dimensional liquidchromatography and tandem mass spectrometry. 1% FPR^(a) 5% FPR^(a) #MS/MS spectra identified^(b) 5915 12819 # Unique sequences^(c) 2731 5601# Unique vaccinia sequences^(d) 65 116 # Vaccinia proteinsrepresented^(e) 44 61 ^(a)Database search results summarized at the 1%false positive rate (FPR) and the 5% FPR. ^(b)Number of tandem massspectrometry (MS/MS) search results surpassing scoring thresholds thatcharacterize the 1% and 5% FPR. Includes results for polypeptidesidentified in multiple strong cation exchange fractions, multiple MS/MSspectra from the same precursor m/z, at multiple charge states, and frommultiple database entries containing the identified sequence. ^(c)Numberof unique polypeptide sequences identified, from all species representedin the database (human, bovine, vaccinia proteins). ^(d)Number ofpolypeptide sequences identified that are unique to vaccinia proteins.^(e)Number of vaccinia proteins represented by the identified vacciniapolypeptides.

The naturally processed and presented vaccinia polypeptides identifiedwere listed in Table 2 and Table 3. The polypeptides were sorted by theopen reading frame (ORF) they originated from and were marked whetherthey were identified within database search scoring criteria thatcharacterized the 5% FPR (italicized) or the 1% FPR (non-italicized).Vaccinia polypeptide sequences identified at the 5% or better FPR wereselected for additional studies to characterize their immunogenicproperties.

TABLE 2Class I polypeptides from Vaccinia virus identified by two-dimensional liquidchromatography and tandem mass spectrometry after Vaccinia infection of human B-cells.SEQ CBS ID Polypeptide Vaccinia Putative IC50 NO. sequence^(a) ORFstrain^(b) Other pox viruses^(c) allele^(d) BIMAS^(e) (nM)^(f)SYFPEITHI^(g) 1 ILIRGIINV A C, V None A*0201 271.9 26 30 ORF T 2AQITTDDLVKSY A10L C, V Vr, Cw, Mn, Ra B*1501 3 VQAVTNAGKIVY A12L A, C, VVr, Cm, Cw, Mn, Ra B*1501 4

A1L A, C, V Cm, Cw A*0201 5 LLFEDIIQNEY A23R A, C, T, V Vr, Cm, Cw, MnB*1501 114 6 FTVNIFKEV A24R A, C, T, V Vr, Cm, Cw, Mn, Ra A*0201 8.4 19516 7 GDKFTTRTSQKGTVAY A24R A, C, T, V Vr, Cm, Cw, Mn, Ra B*1501 8ILYDPETDKPY A24R A, C, T, V Vr, Cm, Cw, Fw, Mn, Ra B*1501 81 9TTRTSQKGTVAY A24R A, C, T, V Vr, Cm, Cw, Mn, Ra B*1501 10 VIINSTSIF A24RA, C, T, V Vr, Cm, Cw, Mn, Ra B*1501 10.0 223 13 11 LTREMGFLVY A29RA, C, T, V Vr, Cm, Cw, Mn, Ra B*1501 17.4 132 15 12 TVINEDIVSKLTF A29RA, C, T, V Vr, Cm, Cw, Mn, Ra B*1501 13 TLRFLEKTSF A31R C, VVr, Cm, Cw, Mn B*1501 72.0 507 11 14 VQIDVRDIKY A35R C, V Cm, Cw B*105152.8 131 23 15 YIIGNIKTV A35R C, V Cm, Cw Ra A*0201 101.2 143 29 16IQYPGSEIKGNAY A44L A, V Cm, Cw B*1501 17 IQYPGSKIKGNAY A44L C Ra B*150118 IQYPGSKIKGNAYF A44L C Ra B*1501 19 KISNTTFEV A44L A, C, VCm, Cw, Mn, Ra A*0201 194.1 10 23 20 LLISADDVQEIRV A44L A, C, VVr, Cm, Cw, Mn, Ra A*0201 21 TLYDISPGHVYA A44L A, C, V Cm, Cw, Mn, Ra NA22 YPGSKIKGNAY A44L C Ra B*1501 8,183 23 VIRNEVNDTHY A46R C, VVr, Cm, Cw, Ra B*1501 476 24 FQQKVLQEY A48R A, C, T, VVr, Cm, Cw, Mn, Ra B*1501 160.0 112 21 25 VAYAAAKGASM A48R A, C, T, VCm, Cw, Ra NA 14,028 26 I^(ox)MNNPDFKTTY^(h) A49R C, V Vr, Cw B*1501 9727 VQKQDIVKLTVY A52R C, V Cw B*1501 28 KLFNEDLSSKY A7L A, C, T, VVr, Cw, Mn B*1501 183 29 LIQEIVHEV A7L A, C, T, V Vr, Cm, Cw, Mn A*0201153.3 18 29 30 LVIENDSQF A8R A, C, V Vr, Cm, Cw B*1501 1.1 215 19 31KLYKSGNSHIDY B12R A, C, V Cw, Ra B*1501 32 RVFAPKDTESVF B12R A, C, VCw, Ra B*1501 33 KVSAQNISF B13R C, V Vr, Cm, Cw, Mn, Ra B*1501 2.4 117 734 GQLYSTLLSF B15R C, V Vr, Cm, Ra B*1501 96.0 106 21 35 LQYAPRELLQY B1RA, C, V Vr, Cm, Cw, Mn B*1501 78 36

B21R, C15L C Cw NA 39,041 37

C11R A, T None A*0201 83.5 4 27 38 KIKDDFQTVNF C12L C, V Vr, Cm, Cw, MnB*1501 731 39 KIYGSDSIEF C12L C, V Vr, Cw Mn B*1501 14.4 229 12 40

C2L C, T, V Cm, Cw, Ra B*1501 6.3 68 23 41 KLSDSKITV D13L A, C, T, VVr, Cm, Cw, Mn A*0201 998.1 21 24 42 VLSLELPEV D13L A, C, T, VVr, Cm, Cw, Mn A*0201 271.9 14 28 43 ILVPNINILKI D6R A, C, T, VVr, Cm, Cw, Mn NA 78 44

D6R A, C, T, V Vr, Cm, Cw, Fw, Mn, Ra A*0201 46 45 RLKPLDIHY D8LA, C, T, V Cm, Cw, Ra B*1501 172.8 135 23 46 YAIDVSKVKPL E10R CVr, Cm, Cw, Mn A*0201 1,337 47 GKASQNPSK^(ox)MVY E5R C, D Cw, Ra B*150148

E5R C, D Cw, Ra B*1501 49 KLFSDISAI E5R C, D, V Vr, Cm, Cw, Ra NA 310.78 25 50

E5R C, D Cw, Ra B*1501 52.8 88 22 51 LARLGLVL E6R C, VVr, Cm, Cw, Mn, Ra A*0201 52 GSFSGRYVSY E8R C, V Vr, Cm, Cw, Mn, RaB*1501 1.2 204 15 53 KQKFPYEGGKVF E9L A, C, V Vr, Cm, Cw, Ra B*1501 54IQHRQQLELAY F11L C, P Ra B*1501 83 55 IQKDINITHY F11L C, PVr, Cm, Cw, Ra NA 0.0 42,108 4 56 MLTEFLHYC F11L C, P Vr, Cw, Mn, Ra NA1,664,5 105 17 57 LF^(ox)MDEIDHESY^(h) F12L C, P Vr, Cm, Cw, Mn, RaB*1501 566 58 VQILMKTANNY F12L C Vr, Cm B*1501 106 59 KQISISTGVLY F16L CVr, Cm, Cw, Mn B*1501 85 60 RVKQISISTGVLY F16L C Vr, Cm, Cw, Mn B*150161

F1L A, C, P, T, V Cw A*0201 62 RQLPTKTRSY F1L A, C, P, T, VVr, Cm, Cw, Mn, Ra B*1501 96.0 80 25 63 ILKSEIEKATY G4L C, V Cw B*1501374 64 ILIEIIPKI H4L A, C, V Vr, Cm, Cw, Mn, Ra NA 167.2 4 31 65ITNKADTSSF H5R C, V Vr B*1501 2.6 276 10 66 IIKEDISEY H7R A, C, T, VVr, Cm, Cw, Mn, Ra B*1501 42.9 171 19 67 YSKKFQESF I1L A, C, P, T, VCm, Cw, Mn B*1501 6.0 185 11 68 KLLLGELFFL J3R A, C, V Vr, Cm, Cw, MnA*0201 20,297.3 7 27 69 LQKGHNKFPVNK J4R C, V Vr, Cm, Cw, Mn B*1501 70VVIGNTLIKY J6R A, C, V Vr, Cw, Mn, Ra B*1501 2.9 242 22 71

K1L C, V None B*1501 2.4 103 16 72

K1L C, V None B*1501 13.2 154 18 73 SLLFIPDIKL K1L C, V Vr, Cw, Mn, RaA*0201 79.0 61 25 74 SQFDDKQNTALY K1L C, V Cm, Cw Mn, Ra B*1501 75VLLDDAEIAK^(ox)M K1L V Cm, Cw, Mn, Ra NA 55 76 VLLDDAEIAK^(ox)MII^(h)K1L V Cm, Cw, Mn, Ra NA 77 KLVGKTVKV K3L C, V Cm, Cw, Mn A*0201 243.3 5430 78 ITYPKALVF K6L C, V Cw, Mn B*1501 4.1 168 11 79 MMIDDFGTARGNY K6LC, V None B*1501 80

K7R A, C, V Cw B*1501 147 81 RLYKEL^(ox)KKF^(h) K7R A, C, V Cm, Cw, RaB*1501 40.0 763 20 82 HIIKEFMTY N2L C, V Vr, Cm, Cw, Mn, Ra B*1501 11.0797 18 83 SIIAILDRF N2L V Vr, Cm, Cw, Ra B*1501 20.0 3,775 16

TABLE 3Class I polypeptides from vaccinia virus identified by two-dimensional liquidchromatography and tandem mass spectrometry after vaccinia infection of human B-cells.SEQ CBS ID Polypeptide Vaccinia Putative IC50 NO: sequence^(a) ORFstrain^(b) Other pox viruses^(c) allele^(d) BIMAS^(e) (nM)^(f)SYFPEITHI^(g) 84 GLLDRLYDL O1L C, V Cm, Cw, Ra A*0201 645.4 10 29 85IVIEAIHTV A48R A, C, T, V Vr, Cm, Cw, Mn, Ra A*0201 97.6 53 27 86ILSDENYLL A6L C, V Vr, Cm, Cw, Mn, Ra A*0201 148.9 10 24 87 ILDDNLYKVG5R C Vr, Cm, Cw, Mn, Ra A*0201 446.0 4 30 88 KLFTHDIML D12L A, C, VVr, Cm, Mn A*0201 276.6 15 22 89 KIDYYIPYV E2L C, V Vr, Cm, Cw, Mn, RaA*0201 169.4 2 24 90 FLTSVINRV F12L C Vr, Cm, Cw, Mn, Ra A*0201 735.9 525 91 NLFDIPLLTV F12L C, P Vr, Cm, Cw, Mn, Ra A*0201 2,426.7 7 29 92

A, C, V Vr, Cm, Cw, Mn B*1501 288.0 65 23 93 SQIFNIISY A17L C, VVr, Cm, Cw, Mn B*1501 96.0 74 9 94 ALDEKLFLI A23R A, C, T, VVr, Cm, Cw, Mn NA 228.2 6 27 95 HMIDKLFYV A23R A, C, T, V Vr, Cm, Cw, MnA*0201 513.8 2 26 96 QIDVRDIKY A35R C, V Cm, Cw B*1501 1.8 12,810 16 97SIMDFIGPYI A35R C, V Cm, Cw, Mn, Ra NA 119.7 11 21 98 YAAAKGASM A48RA, C, T, V Cm, Cw, Ra NA 0.3 4,895 17 99 ILQNRLVYV A52R C, V Cw A*02011,495.7 13 28 100 IQFMHEQGY B1R A, C, V Vr, Cm, Cw, Mn B*1501 52.0 10621 101 TLLDHIRTA B22R, C16L C Cw, Ra NA 34.7 84 23 102 RQFYNANVL C2LC, T, V Cm, Cw, Ra A*0201 6,960 11 103 ILKINSVKY D12L A, C, V Vr, Cm, MnB*1501 187.2 309 22 104 LLLETKTILV E9L A, C, V Vr, Cm, Cw, Mn, Ra A*02011,793.7 11 26 105 SLSNLDFRL F11L C, P Cw, Mn, Ra A*0201 123.9 30 23 106

F1L A, C, P, T, Y Cw A*0201 1,793.7 8 26 107 QLIYQRIYY F2L C, P, VVr, Cm, Cw, Mn, Ra B*1501 26.4 250 21 108 SLKDVLVSV G5.5E A, C, T, VVr, Mn A*0201 23.0 18 30 109

G5R C Vr, Cm, Cw, Mn, Ra B*1501 2.8 18,814 12 110 NTIDKSSPL I1LA, C, T, V Mn A*0201 1.2 3,795 18 111 TQFNFNGHTY I1L A, C, P, T, VCm, Cw, Mn B*1501 88.0 70 22 112

J6R A, C, V Vr, Cm, Cw, Mn, Ra B*1501 2.6 49 11 113 YLFDYPHFEA K3L VNone NA 2,010.7 8 20 114 IINKDGKQY M1L C, V Vr, Cw, Mn, Ra B*1501 14.3423 18 115 QAIEPSGNNY N1L C, V Cw, Ra B*1501 2.2 146 12 116 ILFRMIETYN1L C, V Vr, Cw B*1501 57.2 208 24

FIG. 2 shows the distribution of polypeptide lengths and putative allelefrom the Vaccinia subset (FIG. 2A) as well as all class I polypeptidesidentified at the 5% FPR rate (FIG. 2B). The majority of thepolypeptides were 9-mers in both the full set of polypeptides as well asthe Vaccinia subset. However, there were a significant number ofpolypeptides longer than 11 amino acids in the full set of identifiedpolypeptides as well as in the Vaccinia subset. Since the W6/32 antibodyused to isolate the HLA-polypeptide complexes had affinity for each ofthe major HLA class I alleles, the list of identified polypeptidesreflected the allotypes of the host cell line; in this case HLA-A*0201,B*1501, and C*03. As a first approximation, identified polypeptides wereassociated with an allele by comparing the C-terminal amino acid to thereported binding motifs at the P9 position (Rammensee et al.,Immunogenetics, 41:178-228 (1995)). Polypeptides terminating in L or Vwere assigned to A*0201, and polypeptides terminating in F or Y wereassigned to B*1501, while the remaining polypeptides were designated as“not assigned.” FIGS. 2C and 2D shows an estimate of the distribution ofpolypeptides among the A and B alleles, and while the method ofassociating a polypeptide with its putative allele was rudimentary, itclearly showed a significant subset of the polypeptides being presentedby the B allele. There was also an unknown contribution of polypeptidesfrom the HLA-C allele, although the density of HLA-C molecules on thesurface of cells has been reported as being 6-fold less than that of theA and B alleles (Snary et al., Eur. J. Immunol., 7(8):580-585 (1977) andNeisig et al., J. Immunol., 160(1):171-179 (1998)).

The vaccinia polypeptides identified also beared evidence of the geneticheterogeneity of the Dryvax strain (Osborne et al., Vaccine,25(52):8807-8832 (2007)). For example, two forms of the same polypeptidefrom ORF A44L were identified: IQYPGSKIKGNAY (SEQ ID NO:17) from theCopenhagen strain, and IQYPGSEIKGNAY (SEQ ID NO:16) from the WesternReserve and Ankara strains of vaccinia. At first glance, these twosequences were flagged as redundant identities of the same polypeptide,since they varied only in one amino acid residue resulting in a massdifference of one Dalton. However, these sequences were assigned fromtwo distinctly different polypeptides with IQYPGSEIKGNAY (SEQ ID NO:16)identified from SCX fractions 4 and 5, and IQYPGSKIKGNAY (SEQ ID NO:17)identified from SCX fractions 14-16. FIGS. 3A and 3B shows the annotatedMS/MS spectra for the two polypeptides. Orbitrap spectra for eachprecursor mass (inset) illustrated the difference in mass for the twopolypeptides (0.5 on the m/z axis, where z=2 as shown by the isotopespacing). Fragment ions were observed from the N-terminal end of thepolypeptide (b-ions, highlighted in dark gray) and from the C-terminalend of the polypeptide (y-ions, highlighted in light gray), while the y-and b-ions highlighted within the dashed box delineated the sequencedifference between the two polypeptides. Table 2 and Table 3 containsother instances where polypeptides were identified that were unique tospecific Vaccinia strains.

Comparison of Directly Identified Vaccinia Class I Polypeptides toPredictive Algorithms

The Vaccinia protein sequences represented by this set of polypeptideswere submitted to three algorithms for predicting potential epitopesaccording to their predicted alleles. Results from the NetMHC (Buus etal., Tissue Antigens, 62(5):378-384 (2003) and Nielsen et al., ProteinSci., 12(5):1007-1017 (2003)), and BIMAS (Parker et al., J. Immunol.,152:163-175 (1994)) and SYFPEITHI (Rammensee et al., Immunogenetics,50:213-219 (1999)) algorithms are shown in Table 3. The NetMHC algorithmused a neural network approach to determine likely epitopes from proteinsequence, calculated a predicted binding affinity to HLA molecules, andclassified this binding as being strong (IC₅₀<50 nM), weak (IC₅₀>50 nMand <500 nM), or less than weak (IC₅₀>500 nM)(http://www.cbs.dtu.dk/services/NetMHC/, accessed Jan. 4, 2008). FIG. 4summarizes how the polypeptides that were directly identified by MS werepredicted by the NetMHC algorithm to bind with HLA molecules.Twenty-seven (22%) of the Vaccinia polypeptides identified by MS werepredicted by the algorithm to be strong binders with another 49 (41%)predicted to be weak binding. Twenty-four polypeptides identified byMS/MS were not predicted by the algorithm, 23 because of excess length.

Additionally, a comparison was made of the list of identified Vacciniapolypeptides to Vaccinia epitopes, from all alleles, contained in theImmune Epitope Database and Analysis Resource (IEDB,http://www.immuneepitope.org/home.do, accessed Jan. 4, 2008) (Peters etal., Nat. Rev. Immunol., 7(6):485-490 (2007)). From the list of 116directly identified polypeptides, 23 were an exact match to an IEDBdatabase record, while another 7 polypeptides were contained within aVaccinia epitope from the IEDB database. These results demonstrated boththe complementary information provided by direct identification ofepitopes by MS, and the limitations inherent in relying solely oncomputer algorithms for understanding the spectrum of polypeptidespresented by natural infection.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An isolated polypeptide, wherein the amino acid sequence of saidpolypeptide is as set forth in any one of SEQ ID NOs:1-83.
 2. Acomposition comprising at least one isolated polypeptide selected fromthe group consisting of SEQ ID NOs:1-82 and 83 and at least onepolypeptide selected from the group consisting of SEQ ID NOs:84-115 and116.
 3. The composition of claim 2, wherein said composition furthercomprises an adjuvant.
 4. A method of preventing or treating variolavirus infection in a subject, wherein said method comprisesadministering to said subject a composition comprising an adjuvant andat least one polypeptide, wherein the amino acid sequence of saidpolypeptide is as set forth in any one of SEQ ID NOs:1-83.
 5. The methodof claim 4, wherein said subject is a human.
 6. A vaccine comprising atleast one isolated polypeptide, wherein the amino acid sequence of saidpolypeptide is as set forth in any one of SEQ ID NOs:1-83.
 7. Thevaccine of claim 6, wherein said vaccine comprises at least onepolypeptide selected from the group consisting of SEQ ID NOs:84-115 and116.
 8. The vaccine of claim 6, wherein said vaccine comprises anadjuvant.
 9. A method of enhancing the immune response in a subject to avaccine, wherein said method comprises administering an agent capable ofincreasing the expression of a transporter associated with antigenprocessing in said subject, wherein said vaccine comprises at least oneisolated polypeptide, wherein the amino acid sequence of saidpolypeptide is as set forth in any one of SEQ ID NOs:1-83.
 10. Themethod of claim 9, wherein said transporter is TAP1, TAP2, or Tapasin.11. A method of inducing an immune response against at least oneisolated polypeptide selected from the group consisting of SEQ IDNOs:1-82 and 83, wherein said method comprises administering saidpolypeptide to a subject in an amount effective to induce an immuneresponse against said polypeptide.
 12. The method of claim 11, whereinsaid polypeptide is administered in combination with a polypeptideselected from the group consisting of SEQ ID NOs: 84-115 and
 116. 13.The method of claim 11, wherein said polypeptide is administered incombination with a pharmaceutically acceptable excipient, carrier,diluent, or vehicle.
 14. The method of claim 11, wherein said methodcomprises administering to said subject an agent capable of increasingexpression of a TAP molecule.
 15. The method of claim 11, wherein saidimmune response is a cell mediated immune response.
 16. The method ofclaim 15, wherein said cell mediated immune response is a cell mediatedcytolytic immune response.
 17. The method of claim 15, wherein said cellmediated immune response is a class I-restricted T cell response.
 18. Akit comprising (a) at least one polypeptide selected from the groupconsisting of SEQ ID NOs:1-82 and 83, and (b) an adjuvant.
 19. The kitof claim 18, wherein said kit comprises at least two polypeptidesselected from said group.
 20. The kit of claim 18, wherein said kitcomprises at least one polypeptide selected from the group consisting ofSEQ ID NOs:84-115 and 116.